Comparison of Two Site-Specifically 18F-Labeled Affibodies for PET

Subscriber access provided by Maastricht University Library

Article

Comparison of Two Site-Specifically 18F-Labeled Affibodies for PET Imaging of EGFR Positive Tumors Xinhui Su, Kai Cheng, Jongho Jeon, Bin Shen, Gianina Teribele Venturin, Xiang Hu, Jianghong Rao, Frederick T Chin, Hua Wu, and Zhen Cheng Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp5003043 • Publication Date (Web): 27 Jun 2014 Downloaded from http://pubs.acs.org on July 4, 2014

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Comparison of Two Site-Specifically 18F-Labeled Affibodies for PET Imaging of EGFR Positive Tumors Xinhui Su†,‡, Kai Cheng‡, Jongho Jeon2‡, Bin Shen‡, Gianina Teribele Venturin‡,§, Xiang Hu‡, Jianghong Rao‡, Frederick T. Chin‡, Hua Wuǁ , and Zhen Cheng*,‡

† Department of Nuclear Medicine, Zhongshan Hospital Xiamen University, Xiamen 361004, China ‡ Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Bio-X Program and Stanford Cancer Center, Stanford University School of Medicine, Stanford, CA 94305, USA § Universidade Católica do Rio Grande do Sul (PUCRS) – Porto Alegre – Brazil. ǁ Department of Nuclear Medicine, The First Affiliated Hospital of Xiamen University, Xiamen 361003, China

Corresponding Author Zhen Cheng, Ph.D. Molecular Imaging Program at Stanford, Department of Radiology and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, 1201 Welch Road, Lucas Expansion, P095 Stanford University Stanford, CA 94305-5484. Phone: 650-723-7866. Fax: 650-736-7925. E-mail: [email protected].

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 37

Abstract The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous PET studies showed that the EGFR-targeting Affibody molecules 64Cu-DOTA-ZEGFR:1907 and 18F-FBEM-ZEGFR:1907 can discriminate between high and low EGFR-expression tumors, and have the potential for patient selection for EGFR-targeted therapy. Compared with

64

Cu,

18

F may improve

imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of

18

study

to

is

F-FBEM-ZEGFR:1907 requires a long synthesis time. The aim of the present develop

a

new

(Al18F-NOTA-ZEGFR:1907 and

18

generation

of

18

F

labeled

Affibody

probes

F-CBT-ZEGFR:1907) and to determine whether they are

suitable agents for imaging of EGFR expression. Methods: The first approach consisted of conjugating ZEGFR:1907 with NOTA and radiolabeling with Al18F to produce Al18F-NOTA-ZEGFR:1907. In a second approach the prosthetic group cyanobenzothiazole

(18F-CBT)

was

conjugated

to

Cys-ZEGFR:1907

18

F-labeled-2-

to

produce

18

F-CBT-ZEGFR:1907. Binding affinity and specificity of Al18F-NOTA-ZEGFR:1907 and

18

F-CBT-ZEGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET

studies were conducted on mice bearing A431 xenografts after injection of Al18F-NOTA-ZEGFR:1907 or

18

F-CBT-ZEGFR:1907 with or without co-injection of unlabeled

Affibody proteins. Results: The radiosyntheses of Al18F-NOTA-ZEGFR:1907 and 18

F-CBT-ZEGFR:1907 were completed successfully within 40 and 120 min with a

decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 2


Page 3 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


cells. Although

18

F-CBT-ZEGFR:1907 showed instability in vivo, biodistribution studies

revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al18F-NOTA-ZEGFR:1907 demonstrated high in vitro and in vivo stability, high tumor uptake and relative low uptake in most of the normal organs except the liver and kidneys at 3 h after injection. The specificity of both probes for A431 tumors was confirmed by their lower uptake on co-injection of unlabeled Affibody. PET studies showed that Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 could clearly identify EGFR-positive tumors with good contrast. Conclusion: Two strategies for 18F-labeling of Affibody molecules were successfully developed as two model platforms using NOTA or CBT coupling

to Affibody

Al18F-NOTA-ZEGFR:1907 and

18

molecules that

contain an

N-terminal cysteine.

F-CBT-ZEGFR:1907 can be reliably obtained in a relatively

short time. Biodistribution and PET studies demonstrated that Al18F-NOTA-ZEGFR:1907 is a promising PET probe for imaging EGFR expression in living mice. Key Words: Affibody; EGFR; PET; 18F; NOTA; CBT

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Introduction The epidermal growth factor receptor (EGFR) plays an important role in neoplastic processes of cell proliferation, inhibition of apoptosis, angiogenesis, and metastatic spread (1). Overexpression of EGFR in tumors has been associated with resistance against conventional drug treatment and radiation and may predict poor prognosis (2-4). Detection of EGFR expression by molecular imaging could be a useful tool for evaluation of antitumor drug effect, stratification of cancer patients for molecularly targeted therapy, and prognosis of cancer patients, as it could provide real time data with fewer false-negative results (5). Affibody molecules are based on a 58 amino acid residue protein domain, derived from one of the IgG-binding domains of staphylococcal protein A, and has been engineered to be chemically stable and to bind target proteins with high affinity (6, 7). Because of their small size (~7 kDa) and high affinity, Affibody molecules generally show fast and good tumor tissue penetration and accumulation, and rapid clearance from the blood, resulting in high imaging contrast within a short period (for example: 0.5-1 h) after injection. Anti-human epidermal growth factor receptor 2 (HER2) Affibody molecules (ZHER2) and their derivatives have been radiolabeled with various radionuclides for imaging of tumors overexpressing HER2 in animal models (7-10). Subsequently, 111

In- or

68

Ga-labeled ZHER2 have been successfully and safely used to visualize

HER2-expressing tumors in patients with metastatic breast cancer. These clinical studies clearly demonstrate that Affibody molecules have great potential to become a promising new class of cancer-targeting ligands for clinical translation (11). Overall the previous 4


Page 4 of 37

Page 5 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


preclinical and clinical studies encourage us to further develop clinical translatable Afffibody probes to image other tumor targets such as EGFR (12,13). We have previously reported the site-specific coupling of an anti-EGFR Affibody molecule (ZEGFR:1907) with maleimido-mono-amide-DOTA (MMA-DOTA, 1,4,7,10tetraazacyclododecane-1,4,7-trisaceticacid-10-maleimidoethylmonoamide) to produce the bioconjugate, DOTA-ZEGFR:1907, that was radiolabeled with

64

Cu (13). This conjugate

allowed high-contrast imaging of EGFR-expressing xenografts. However, imaging of EGFR expression with Affibody molecules and further clinical translation of them can be further improved by

18

F-labeling. Not only are

18

F probes more clinically relevant than

64

Cu, but also they have good imaging characteristics and a suitable half-life for relatively

low molecular weight proteins and peptides. Therefore we recently radiolabeled ZEGFR:1907 with N-2-(4-18F-fluorobenzamido)ethyl maleimide (18F-FBEM) to produce the PET probe, 18

F-FBEM-ZEGFR:1907, for imaging EGFR expression in a variety of tumor models (12).

Although 18F-FBEM-ZEGFR:1907 PET allowed us to visualize EGFR-expressing tumors, the labeling procedure to obtain the probe is complex and tedious, and requires a long radiosynthesis time (4-step radiosynthesis, 3 h, 10% decay corrected yield), which severely limits further applications of 18F-FBEM-ZEGFR:1907. Recently, two new and simple methods for labeling of biomolecules with been

developed.

In

the

first

one,

peptides

conjugated

to

18

F have

MMA-NOTA

(1,4,7-triazacyclononane-N,N’,N’’-triacetic acid maleimidoethylmonoamide) and its analogues have been labeled with 18F via the formation of aluminum 18F-fluoride (Al18F) and its complexation by NOTA directly (one step radiosynthesis) (14, 15). The second 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 37

method involves 18F-labeling of N-terminal cysteine-bearing peptides and proteins and is based on a rapid condensation reaction between

18

F-fluorinated-2- cyanobenzothiazole

(18F-CBT) and cysteine (2-step reaction) (16). Both methods allow rapid and efficient labeling of peptides and proteins with 18F. Al18F-NOTA in particular has been applied to label many peptides including RGD and anti-HER2 Affibody molecules (17, 18). The Al18F-NOTA labeled RGD peptides have also been successfully used for PET imaging of a lung cancer patient recently (19). Our ultimate goal is to translate an

18

F-labeled ZEGFR into clinical applications.

Therefore in the current study, we aimed to use the above radiofluorination strategies (Al18F-NOTA, 18F-CBT) to site-specifically label ZEGFR:1907 and further determine whether the resulting PET probes, Al18F-NOTA-ZEGFR:1907 and

18

F-CBT-ZEGFR:1907, are suitable

agents for imaging mice bearing EGFR expressing A431 tumors. For this purpose, NOTA-conjugated ZEGFR:1907 was prepared and radiolabeled with

18

F to produce

Al18F-NOTA-ZEGFR:1907, and the prosthetic group (18F-CBT) was conjugated to Cys-ZEGFR:1907 to produce

18

F-CBT-ZEGFR:1907 (Figure 1). The in vitro properties and in

vivo performance of Al18F-NOTA-ZEGFR:1907 were then compared with those of 18

F-CBT-ZEGFR:1907 in A431 cells and tumor xenografts.

MATERIALS AND METHODS General MMA-NOTA was purchased from CheMatech Inc. (Dijon, France). Phosphatebuffered saline (PBS), high-glucose dulbecco's modified eagle medium (DMEM), 10% 6


Page 7 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


fetal bovine serum (FBS), 1% penicillin-streptomycin, 0.1% trypsin, trypsin-EDTA and TrypLE-Express were purchased from Invitrogen Life Technologies (Carlsbad, California). Dimethylsulfoxide (DMSO) and acetonitrile (MeCN) were purchased from Fisher Scientific (Pittsburgh, Pennsylvania). Dimethylformamide (DMF), trifluoroacetic acid (TFA), thioanisole (TIS), ethanedithiol (EDT), ethylene-diamine-tetra-acetic acid (EDTA),

tris(2-carboxyethyl)-phosphine

hydrochloride

(TCEP

HCl),

N,N-diisopropyl-ethylamine (DIPEA), ethyl acetate, dithiothreitol (DTT), mouse serum and all other standard synthesis reagents were purchased from Sigma-Aldrich Chemical Co.(St. Louis, Missouri). All chemicals were used without further purification. The Affibody molecules Ac-Cys-ZEGFR:1907 (Ac-CVDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQAPK-NH2) ZEGFR:1907

and

Cys-

(CVDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPSQSA-

NLLAEAKKLNDAQAPK-NH2) were synthesized on a CS Bio CS336 instrument (CS Bio Company, Menlo Park, California) in our laboratory as previously described (13). The purified peptide was dissolved in water, and the concentration was determined by amino acid analysis (Molecular Structure Facility, University of California, Davis, CA). Peptide purity and molecular mass were determined by analytic scale reversed-phase high-performance liquid chromatography (RP-HPLC, model: 3000 HPLC System, Dionex

Corporation,

Sunnyvale,

California)

and

matrix-assisted

laser

desorption/ionization–time of flight mass spectrometry (MALDI-TOF-MS, model: Perseptive

Voyager-DE

RP

Biospectrometer,

Framinghan,

Massachusetts),

or

electrospray ionization mass spectrometry (ESI-MS, model: Micromass ZQ single 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 37

quadrupole LC-MS, Milford, Massachusetts) as previously described (13). The human epidermoid carcinoma cancer cell line A431 was obtained from the American Type Tissue Culture Collection (Manassas, Virginia). Female nude mice were purchased from Charles River Laboratories (Boston, Massachusetts). Radiosynthesis of Al18F-NOTA-ZEGFR:1907 The Affibody molecule NOTA-ZEGFR:1907 was radiolabeled with

18

F according to a

previously reported method (14,15,18) (Figure 1A). First, Ac-Cys-ZEGFR:1907 was conjugated with the bifunctional chelator MMA-NOTA using the method described below: Ac-Cys-ZEGFR:1907 was dissolved in freshly degassed phosphate buffer (0.1 M, pH 7.4) at a concentration of 1 mg/mL. Twenty equivalents of MMA-NOTA dissolved in DMSO (10 mM) were added. After mixing by vortexing for 2 h, the product was purified by RP-HPLC with a protein-and-peptide C4 column (Grace Vydac 214TP54, Columbia, Maryland) using a gradient system of solvent A (0.1% TFA/H2O) and solvent B (0.1%TFA/MeCN). The flow rate was 4 mL/min, with the mobile phase starting from 90% solvent A and 10% solvent B (0-3 min) to 35% solvent A and 65% solvent B at 33 min. Fractions containing the product were collected and lyophilized. The identity of the products was confirmed by MALDI-TOF-MS. Second,

nonradioactive

Al19F-NOTA-ZEGFR:1907, a

reference

standard,

was

synthesized with NOTA-ZEGFR:1907 and K19F. To a solution of KF (2 mM, 5 µL) in 20 µL of sodium acetate buffer (0.1 M, pH 4) was added AlCl3 (2 mM, 5 µL). Then, NOTA-ZEGFR:1907 (50 µg), dissolved in 50 µL of sodium acetate buffer (0.1 M, pH 4) was added and the reaction mixture was incubated for 15 min at 100 °C. The resulting 8


Page 9 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


conjugate, Al19F-NOTA-ZEGFR:1907 was purified by HPLC. Lastly,

18

F radiolabeling of NOTA-ZEGFR:1907 was performed.

18

F-fluoride (37 x 103

MBq) was prepared by proton bombardment of 2.5 mL of [18O] enriched water target via the

18

O (p, n)

18

F nuclear reaction. The

18

F-fluoride was then trapped onto a Sep-Pak

QMA cartridge (Waters, Milford, Massachusetts), washed with 3 mL of metal-free water and eluted from the cartridge with 100 µL of 0.9% NaCl. Al18F was prepared by adding AlCl3 (2 mM, 2 µL) in sodium acetate buffer (0.1 M, pH 4). NOTA-ZEGFR:1907 (150 µg) was dissolved in 25 µL of sodium acetate buffer (0.5 M, pH 4). To the dissolved Affibody molecule, acetonitrile (25 µL) and Al18F (50 µL, 1.3-1.6 × 103 MBq) were added, then the reaction mixture was incubated for 15 min at 100 oC. An Oasis HLB cartridge (30 mg; Waters) was used to remove unincorporated Al18F, and the desired product was purified with HPLC using the same elution gradient described for NOTA-ZEGFR:1907 purification. The HPLC fractions containing Al18F-NOTA-ZEGFR:1907 were collected, combined, and evaporated. Al18F-NOTA-ZEGFR:1907 was reconstituted in PBS (0.1 M, pH 7.4) and passed through a 0.22 -µm Millipore filter into a sterile vial for in vitro and animal experiments. Radiosynthesis of 18F-CBT-ZEGFR:1907 Nonradioactive 18

19

F-CBT-ZEGFR:1907 was used as a reference for characterization of

F-CBT-ZEGFR:1907 and prepared by reaction of Cys-ZEGFR:1907 with

19

F-CBT. Briefly,

TCEP HCl solution (2.4 µL, 10 mM) and DIPEA (360 nmol) were added to CysZEGFR:1907 solution (30 µL, 200 µM in DMF) and then the resulting solution was mixed with 19F-CBT solution (1.8 µL, 10 mM, 3 equiv.). The resulting mixture was heated to 60 o

C for 1 h. The crude product was purified with semi-preparative HPLC using 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 37

Phenomenex Gemini column (10 mm x 250 mm, 5 µm) using a linear gradient from deionized water with 0.1% TFA to MeCN with 0.1% TFA: 0-3 min 0-40% (MeCN); 3-35 min 40-100% (MeCN), and the flow rate was 3 mL/min. Cys-ZEGFR:19077 was labeled with described (16) (Figure 1B). First,

18

18

F-CBT according to the procedure we recently

F-labeling of tosylated CBT was performed.

18-Crown-6/K2CO3 solution (1 mL, 15:1 MeCN/H2O, 16.9 mg of 18-Crown-6, 4.4 mg of K2CO3) was used to elute the activity of

18

F-fluoride from QMA cartridge into a dried

glass reactor. The resulting solution was azeotropically dried with sequential MeCN evaporations at 90

o

C. A solution of [2-((2-cyanobenzo[d]thiazol-6-yl)-oxy)ethyl

4-methylbenzenesulfonate] (2 mg in 1 mL of anhydrous MeCN) was added to the reactor and the mixture heated at 90 oC for 10 min. After cooling to 30 oC, HCl (0.05 M, 2.5 mL) was added to quench the reaction and prevent basic hydrolysis of the product

18

F-CBT.

The crude mixture was then purified with a semi-preparative HPLC using the same elution gradient described for

19

F-CBT purification. The collected

18

F-CBT solution was

diluted with H2O (20 mL) and passed through a C18 cartridge. The trapped 18F-CBT was eluted out from the cartridge with Et2O (2.5 mL), and the Et2O was removed by a helium stream. The isolated radiochemical yield of 18F-CBT was ca. 20% (5.18-5.55 x 103 MBq, decay–corrected to end of bombardment). For the radiosynthesis of

18

F-CBT-ZEGFR:1907,

Cys-ZEGFR:1907 (150 µg, 7.5 nmol) was dissolved in PBS buffer (0.1 M, pH 7.4) containing 5 eq of TCEP HCl and 50 eq of NaHCO3. The resulting solution was added to

18

F-CBT

(1.85 x 103 MBq) in DMF (200 µL) at 60 oC. After 20 min, the reaction was quenched with 5% AcOH aqueous solution. The crude product was purified with a semi-preparative 10


Page 11 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


HPLC using Phenomenex Gemini column (10 mm x 250 mm, 5 µm) using a linear gradient from deionized water with 0.1% TFA to MeCN with 0.1% TFA: 0-5 min 0-5% (MeCN); 5-42 min 5-65% (MeCN), and the flow rate was 5 mL/min. Cell Assays Cell uptake and receptor saturation assays were performed as previously described with minor modifications (13). Briefly, the EGFR positive A431 cell line was cultured in high glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C, with the medium changed every two days. A 70-80% confluent monolayer was detached by 0.1% trypsin and dissociated into a single cell suspension for further cell culture. Cell uptake assays. The A431 cells were washed three times with PBS and dissociated with 0.25% trypsin-EDTA. DMEM medium was then added to neutralize trypsin-EDTA. Cells were spun down and re-suspended with serum-free DMEM. cells (0.5×106) were incubated at 37 °C for 0.25 to 2 h with 7.4 × 10-3 MBq Al18F-NOTA-ZEGFR:1907 or 18F-CBT-ZEGFR:1907 in 0.5 mL serum-free DMEM medium. The non-specific binding of Al18F-NOTA-ZEGFR:1907 or

18

F-CBT-ZEGFR:1907 with A431 cells

was determined by co-incubation with 0.6 µM non-radiolabeled NOTA-ZEGFR:1907 or Cys-ZEGFR:1907. The cells were washed three times with 0.01 M PBS (pH 7.4) at room temperature. Cell were then washed three times with chilled PBS and spun down at a speed of 7000-8000 rpm. The cell pellets at the bottom of the tube were spliced, and the radioactivity of the pellets was measured using a γ-counter (PerkinElmer 1470, Waltham, 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 37

Massachusetts). The uptake (counts/min) was normalized to the percentage of binding for analysis using Excel (Microsoft Software Inc., Redmond, Washington). Receptor saturation assays. A431 cells (0.3 x 106) were plated on 6-well plates one day before the experiment. Cells were washed with PBS three times. Serum-free DMEM (1 mL) was added to each well, followed by the addition of either Al18F-NOTA-ZEGFR:1907 (8.9-532.8 x 10-3 MBq, 2-120 nM final concentration) or 18F-CBT-ZEGFR:1907 (8.9-532.8 x 10-3

MBq,

2-120

nM

Al18F-NOTA-ZEGFR:1907 or

final 18

concentration).

The

non-specific

binding

of

F-CBT-ZEGFR:1907 with A431 cells was determined by

co-incubation with 100 times excess of NOTA-ZEGFR:1907 or Cys-ZEGFR:1907. The plates were then put on ice for 2 h, and the cells were washed with cold PBS three times and detached with TrypLE-Express. The radioactivity of the cells was measured using a γ-counter. Specific binding (SB) = Total binding (TB) - Non-specific binding (NSB). The data were analyzed using GraphPad Prism (GraphPad Software, Inc., San Diego, California), and the dissociation constant (KD value) of 18

18

F-NOTA- ZEGFR:1907 and

F-CBT-ZEGFR:1907 were calculated from a 1-site-fit binding curve.

In Vitro and In Vivo Stability In vitro and in vivo stability were determined similarly to the procedures previously described with minor modifications (12, 13). In vitro serum stability assay. Al18F-NOTA-ZEGFR:1907 (1.5-6.7 MBq) or 18

F-CBT-ZEGFR:1907 (2.2-7.4 MBq) was incubated in 0.5 mL of mouse serum for 1 and 2 h

at 37 oC. At each time point, the mixture was precipitated with 300 µL of ethanol and centrifuged at 16,000g for 2 min. The supernatant was transferred to a new Eppendrof 12


Page 13 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tube, and DMF (300 µL) was added to precipitated the residue of serum protein. After centrifugation, the supernatant was acidified with 300 µL of buffer A (water + 0.1% TFA) and filtered using a 0.2 -µm nylon Spin-X column (Corning Inc. Corning, New York). The filtrates were then analyzed by radio-HPLC under conditions identical to the ones used to analyze

the

original

radiolabeled

Al18F-NOTA-ZEGFR:1907 and

18

compound.

The

percentage

of

intact

of

F-CBT-ZEGFR:1907 were determined by quantifying peaks

corresponding to the intact and the degradation products. In vivo stability assay. Two groups of A431 mice (for each group n=3) were injected with Al18F-NOTA- ZEGFR:1907 (5.8 MBq) or 18F-CBT-ZEGFR:1907 (7.4 MBq) via a tail vein and euthanized at 1 h after injection. The tumors were removed and homogenized with DMF (0.5 mL) with 1% Triton X-100 (Sigma-Aldrich). Blood samples were centrifuged immediately after collection to remove the blood cells. The plasma portions were added to DMF (0.5 mL) with 1% Triton X-100. After centrifugation, the supernatant portions were diluted with solution A (99.9% H2O with 0.1% TFA) and centrifuged again at 16,000g for 2 min with a nylon filter. The filtrates were analyzed by radio-HPLC under conditions identical to those used for analyzing the original radiolabeled peptide. Biodistribution Studies The animal procedures were performed according to a protocol approved by the Stanford University Institutional Animal Care and Use Committee. Approximately 5 x 106 cultured A431 cells suspended in PBS were implanted subcutaneously in the right upper or lower shoulders of nude mice. Tumors were allowed to grow to around 0.5-1.0 cm in diameter (10-15 days) and then the tumor-bearing mice underwent in vivo biodistribution 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 37

and imaging studies. For biodistribution studies, A431 tumor-bearing mice (for each group n=4) were injected with 18F-NOTA-ZEGFR:1907 (1.9-2.6 MBq) or 18F-CBT-ZEGFR:1907 (1.48-2.22 MBq) with 30 µg of nonradioactive Ac-Cys-ZEGFR:1907 or Cys-ZEGFR:1907, respectively, through a tail vein. At 3 h after injection, the mice were sacrificed, and tumors and normal tissues of interest were removed and weighed, and their radioactivity was measured in a γ-counter. The radioactivity uptake in the tumor and normal tissues was expressed as a percentage of the injected radioactivity per gram of tissue (%ID/g). In order to study the in vivo EGFR targeting specificity of Al18F-NOTA-ZEGFR:1907 and

18

F-CBT-ZEGFR:1907, unlabeled

Ac-Cys-ZEGFR:1907 or Cys-ZEGFR:1907 protein (300 µg) was co-injected with the corresponding

18

F-labeled ZEGFR:1907 in nude mice bearing A431 tumors (n=4) via a tail

vein, and biodistribution studies were conducted at 3 h after injection. Small-Animal PET Imaging PET imaging of tumor-bearing mice was performed on a microPET R4 rodent model scanner (Siemens Medical Solutions USA, Inc., Malvern, Pennsylvania). The mice bearing A431 tumors (for each group n=4) were injected with Al18F-NOTA-ZEGFR:1907 (1.9-2.6 MBq) or

18

F-CBT-ZEGFR:1907 (1.48-2.22 MBq) spiked with 30 or 300 µg of

non-radioactive Ac-Cys-ZEGFR:1907 or Cys-ZEGFR:1907 through the tail vein. At 1, 2 and 3 h after injection, the mice were anesthetized with 2% isoflurane and placed near the center of the field of view of the microPET scanner in prone position. Three-minute static scans were obtained, and the images were reconstructed by a two-dimensional ordered subsets expectation maximum (OSEM) algorithm. No background correction was performed. 14


Page 15 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Regions of interest (ROIs; 5 pixels for coronal and transaxial slices) were drawn over the tumors on decay-corrected whole-body coronal images. The maximum counts per pixel per minute were obtained from the ROIs and converted to counts per milliliter per minute using a calibration constant. Tissue density was assumed to be 1 g/mL, and the ROIs were converted to counts per gram per minute. Image ROI-derived %ID/g values were determined by dividing counts per gram per minute by the injected dose. No attenuation correction was performed. Statistical Methods Statistical analysis was performed using Student's two-tailed t-test for unpaired data. A 95% confidence level was chosen to determine the significance between groups, with a P value less than 0.05 being indicated as a significant difference.

RESULTS Chemistry and Radiochemisty The Affibody molecules Ac-Cys-ZEGFR:1907 and Cys-ZEGFR:1907 with a cysteine at the N-terminal were successfully synthesized using conventional solid phase peptide synthesis and purified by semi-preparative HPLC. The peptides were generally obtained in 10% yield. The retention time for both on analytical HPLC was 26 min. The purified Ac-Cys-ZEGFR:1907 and Cys-ZEGFR:1907 were characterized by MALDI-TOF-MS. The measured molecular weights (MWs) for both constructs were consistent with the expected MWs (for Ac-Cys-ZEGFR:1907, calculated MW=6690.0 and found MW=6690.7; for 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 37

Cys-ZEGFR:1907, calculated MW=6646.0 and found MW=6645.7). Ac-Cys-ZEGFR:1907 was then conjugated with MMA-NOTA and purified by HPLC. The measured MW of the final

product

(NOTA-ZEGFR:1907)

was

m/z=7112.0

[M+H]+

for

(calculated

MW[M+H]+=7112.6), and the purity of NOTA-ZEGFR:1907 was over 95% (retention time: 29 min).

Lastly,

purified

19

F-NOTA-ZEGFR:1907 and

19

F-CBT-ZEGFR:1907

were also

characterized by MALDI-TOF-MS. The measured MWs for both constructs were consistent with the expected MWs (for 19F-NOTA-ZEGFR:1907, calculated MW=7256.0 and measured MW=7256.6; for

19

F-CBT-ZEGFR:1907, calculated MW=6852.0 and measured

MW=6852.9). The recovery yields of

19

F-NOTA-ZEGFR:1907 and

19

F-CBT-ZEGFR:1907 were

70% and 85%, respectively, after purification (retention time, 29 and 26.4 min). The whole radiosynthesis of For 18

18

18

F-NOTA-ZEGFR:1907 was accomplished within 40 min.

F-CBT-ZEGFR:1907 ， the total radiosynthesis was completed within 120 min.

F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 showed a retention time of 29 and 26.4 min

on HPLC, respectively. Both products were found to be more than 95% radiochemically pure, as determined by analytic HPLC. The overall radiochemical yields with decay correction to the end of synthesis for Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 were 15% and 41%, respectively. The specific activity of Al18F-NOTA-ZEGFR:1907 and 18

F-CBT-ZEGFR:1907 were approximately 1.5 × 103 MBq/µmol and 22.2 × 103 MBq/µmol,

respectively. In Vitro Stability and Metabolite Analysis In vitro stability studies allowed us to observe that more than 90% of Al18F-NOTA-ZEGFR:1907 remained intact during 1 to 2 h of incubation in mouse serum 16


Page 17 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(Figure 2A and 2B). More than 90% of

18

F-CBT-ZEGFR:1907 remained intact after 1 h

incubation in mouse serum, while there was about 75% intact 18F-CBT-ZEGFR:1907 after 2 h of incubation (Figure 2C and 2D). Next, the in vivo stability studies are shown in Figure 3. In plasma and tumor, 90% and 85%, respectively, of Al18F-NOTA-ZEGFR:1907 remained intact (Figure 3A and 3B) at 1 h after injection, indicating excellent stability in vivo. On the other hand,

18

F-CBT-ZEGFR:1907 showed much faster degradation in vivo, with only

40% and 24% of intact tracer product in plasma and tumor, respectively (Figure 3C and 3D). In Vitro Cell Binding Assays Cell uptake levels for Al18F-NOTA-ZEGFR:1907 and 18F-CBT-ZEGFR:1907 are shown in Figures 4A and 4C, respectively. Al18F-NOTA-ZEGFR:1907 quickly accumulated in A431 cells and reached a highest value of 12% of applied activity at 1 h. A similar cell uptake pattern was observed for

18

F-CBT-ZEGFR:1907, but the uptake level was much lower than

that observed for Al18F-NOTA-ZEGFR:1907 at 1 h (7% of applied activity). When both probes were incubated with large excesses of nonradioactive Affibody molecules (Ac-Cys-ZEGFR:1907 or Cys-ZEGFR:1907), their uptake levels in A431 cells were significantly inhibited (P

Comparison of Two Site-Specifically 18F-Labeled Affibodies for PET

Recommend Documents